Making the correct cut

The CRISPR/Cas system is a prokaryotic immune system that targets and cuts out foreign DNA in bacteria. It has been adopted for gene editing because it can be designed to recognize and cut specific locations in the genome. A challenge in developing clinical applications is the potential for off-target effects that could result in DNA cleavage at the wrong locations. Slaymaker et al. used structure-guided engineering to improve the specificity of Streptococcus pyogenes Cas9 (SpCas9). They identified enhanced-specificity variants (eSpCas9) that display reduced off-target cleavage while maintaining robust on-target activity

Abstract

The RNA-guided endonuclease Cas9 is a versatile genome-editing tool with a broad range of applications from therapeutics to functional annotation of genes. Cas9 creates double-strand breaks (DSBs) at targeted genomic loci complementary to a short RNA guide. However, Cas9 can cleave off-target sites that are not fully complementary to the guide, which poses a major challenge for genome editing. Here, we use structure-guided protein engineering to improve the specificity of Streptococcus pyogenes Cas9 (SpCas9). Using targeted deep sequencing and unbiased whole-genome off-target analysis to assess Cas9-mediated DNA cleavage in human cells, we demonstrate that “enhanced specificity” SpCas9 (eSpCas9) variants reduce off-target effects and maintain robust on-target cleavage. Thus, eSpCas9 could be broadly useful for genome-editing applications requiring a high level of specificity.

The RNA-guided endonuclease Cas9 from microbial clustered regularly interspaced short palindromic repeat (CRISPR)–Cas adaptive immune systems is a powerful tool for genome editing in eukaryotic cells (1, 2). However, the nuclease activity of Cas9 can be triggered even when there is imperfect complementarity between the RNA guide sequence and an off-target genomic site, particularly if mismatches are distal to the protospacer adjacent motif (PAM), a short stretch of nucleotides required for target selection (3, 4). These off-target effects pose a challenge for genome-editing applications. Here, we report the structure-guided engineering of Streptococcus pyogenes Cas9 (SpCas9) to improve its DNA targeting specificity.

Several strategies to enhance Cas9 specificity have been reported, including reducing the amount of active Cas9 in the cell (3, 5, 6), using Cas9 nickase mutants to create a pair of juxtaposed single-stranded DNA nicks (7, 8), truncating the guide sequence at the 5′ end (9), and using a pair of catalytically inactive Cas9 nucleases, each fused to a FokI nuclease domain (10, 11). Although each of these approaches reduces off-target mutagenesis, they have a number of limitations: Reducing the amount of Cas9 can decrease on-target cleavage efficiency, double nicking requires the concurrent delivery of two single-guide RNAs (sgRNAs), and truncated guides can increase indel formation at some off-target loci and reduce the number of target sites in the genome (12, 13).

Cas9-mediated DNA cleavage is dependent on DNA strand separation (14, 15). Mismatches between the sgRNA and its DNA target in the first 8 to 12 PAM-proximal nucleotides can eliminate nuclease activity; however, this nuclease activity can be restored by introducing a DNA:DNA mismatch at that location (3, 16–19). We hypothesized that nuclease activity is activated by strand separation and reasoned that by attenuating the helicase activity of Cas9, mismatches between the sgRNA and target DNA are less energetically favorable, resulting in reduced cleavage activity at off-target sites (fig. S1).

The crystal structure of SpCas9 in complex with guide RNA and target DNA (14, 15) provides a basis to improve specificity through rational engineering. The structure reveals a positively charged groove, positioned between the HNH, RuvC, and PAM-interacting domains in SpCas9, that is likely to be involved in stabilizing the nontarget strand of the target DNA (Fig. 1, A and B, and fig. S2). We hypothesized that neutralization of positively charged residues within this nontarget strand groove (nt-groove) could weaken nontarget strand binding and encourage rehybridization between the target and nontarget DNA strands, thereby requiring more stringent Watson-Crick base pairing between the RNA guide and the target DNA strand.

(A) Screen of alanine single mutants for improvement in specificity.The top five specificity-conferring mutants are highlighted in red. (B) Assessment of top single mutants at additional off-target loci. (C) Combination mutants improve specificity comparedwith singlemutants. eSpCas9(1.0) and eSpCas9(1.1) are highlighted in red.

To test this hypothesis, we generated SpCas9 mutants consisting of individual alanine substitutions at 31 positively charged residues within the nt-groove and assessed changes to genome-editing specificity (Fig. 2C; fig. S3, A and B; and fig. S4). Single amino acid mutants were tested for specificity by targeting them to the EMX1(1) target site in human embryonic kidney (HEK) cells using a previously validated guide sequence; indel formation was assessed at the on-target site and three known genomic off-target (OT) sites (3, 4). Five of the 31 single amino acid mutants reduced activity at all three off-target sites by a factor of at least 10 compared with wild-type (WT) SpCas9 while maintaining on-target cleavage efficiency, and six others improved specificity by a factor of 2 to 5. These mutants also exhibited improved specificity when tested on a second locus, VEGFA(1) (Fig. 2B).

Although some single amino acid mutants were more specific than WT SpCas9 when targeting EMX1(1) and VEGFA(1), off-target indels were still detectable (~0.5%) (Fig. 2B). To further improve specificity, we performed combinatorial mutagenesis using the top single amino acid mutants identified in the initial screen. Eight out of 34 combination mutants retained wild-type on-target activity and displayed undetectable off-target indel levels at EMX1(1) OT1, VEGFA(1) OT1, and VEGFA(1) OT2 (Fig. 2C and fig. S3, C and D).

To ensure that the observed decrease in off-target activity was not accompanied by reduced on-target activity, we measured on-target indel formation at 10 target sites in three genomic loci using the top 14 mutants (fig. S5) and ranked these based on a combination of preserved on-target activity and decreased off-target activity. We identified three mutants with both high efficiency (WT levels of on-target indel formation) and specificity SpCas9 (K855A), SpCas9 (K810A/K1003A/R1060A) [also referred to as eSpCas9(1.0)], and SpCas9 (K848A/K1003A/R1060A) [also referred to as eSpCas9(1.1)]. These three variants were selected for further analysis.

We expanded this assay to assess whether SpCas9(K855A), eSpCas9(1.0), and eSpCas9(1.1) broadly retained efficient nuclease activity, measuring on-target indel generation at 24 target sites spanning 10 genomic loci (Fig. 3A). All three mutants generated similar indel levels as WT SpCas9 with the majority of target sites (Fig. 3B). Mutants were expressed equivalently or at higher levels than WT SpCas9 based on a Western blot (Fig. 3C), indicating that improvements in specificity were not due to decreased protein expression levels.

We compared the specificity of the three mutants to WT SpCas9 with truncated guide sequences [18 nucleotides for EMX1(1) and 17 nucleotides for VEGFA(1)], which have been shown to reduce off-target indel formation (12) (fig. S6). When using full-length (20 nucleotides) guides, all three mutants reduced cleavage at all off-target sites assessed compared with WT spCas9. Specifically, eSpCas9(1.0) and eSpCas9(1.1) with 20-nucleotide RNA guides eliminated cleavage at 22 out of 24 off-target sites (<0.2% indel). In contrast, WT SpCas9 with truncated guides eliminated 14 out of 24 sites but also increased off-target activity at five sites compared with WT SpCas9 with 20-nucleotide guides.

To further understand the tolerance SpCas9(K855A), eSpCas9(1.0), and eSpCas9(1.1) for mismatched target sites, we systematically mutated the VEGFA(1) guide sequence to introduce single- and double-base mismatches at different positions (Fig. 4, A to C). Compared with WT SpCas9, all three mutants induced lower levels of indels with mismatched guides. Of note, eSpCas9(1.0) and eSpCas9(1.1) induced lower indel levels even with single-base mismatches located outside of the 7- to 12–base pair seed sequence. Given that we did not observe any difference between eSpCas9(1.0) and eSpCas9(1.1) in terms of specificity, we selected SpCas9(K855A) and eSpCas9(1.1) for further analysis based on on-target efficiency.

We assessed the genome-wide editing specificity of SpCas9(K855A) and eSpCas9(1.1) using BLESS (direct in situ breaks labeling, enrichment on streptavidin and next-generation sequencing) (20, 21), which quantifies DNA double-stranded breaks (DSBs) across the genome (fig. S7A), for both the EMX1(1) and VEGFA(1) targets for both mutants and compared these results to WT SpCas9. We used a previously established computational pipeline for distinguishing Cas9-induced DSBs from background DSBs (21) (fig. S7B). Both SpCas9 (K855A) and eSpCas9(1.1) exhibited a genome-wide reduction in off-target cleavage and did not generate any new off-target sites (Fig. 5, A to D).

These findings also provide insight into the mechanism of Cas9 targeting and nuclease activity. We propose that off-target cutting occurs when the strength of Cas9 binding to the nontarget DNA strand exceeds forces of DNA rehybridization. Consistent with this model, mutations designed to weaken interactions between Cas9 and the noncomplementary DNA (ncDNA) strand led to a substantial improvement in specificity. The model also suggests that, conversely, specificity can be decreased by strengthening the interactions between Cas9 and the nontarget strand. Consistent with this hypothesis, we generated two mutants, S845K and L847R, each of which exhibited decreased specificity (fig. S8). Similar strategies described in this study can also be successfully applied to other Cas9 family proteins, such as Staphylococcus aureus Cas9 (SaCas9) (fig. S9), to engineer nucleases with improved specificity.

We have demonstrated through structure-guided design that neutralization of positive charges in the nt-groove can dramatically decrease off-target indel formation while preserving on-target activity. These data show that eSpCas9(1.1) can be used to increase the specificity of genome-editing applications. Future structure-guided interrogation of Cas9 binding and cleavage mechanism will likely enable further optimization of the CRISPR-Cas9 genome-editing toolbox.

Acknowledgments: We thank J. Dahlman for helpful discussions and a critical review of the manuscript; F. A. Ran, R. J. Platt, and J. Joung for experimental assistance; and the entire Zhang laboratory for support and advice. I.S. is supported by the Simons Center for the Social Brain. W.X.Y. is supported by T32GM007753 from the National Institute of General Medical Sciences and a Paul and Daisy Soros Fellowship. F.Z. is supported by the National Institutes of Health through NIMH (5DP1-MH100706 and 1R01MH110049) and NIDDK (5R01DK097768-03), a Waterman Award from the National Science Foundation, the Keck, New York Stem Cell, Damon Runyon, Searle Scholars, Merkin, and Vallee Foundations, and B. Metcalfe. F.Z. is a New York Stem Cell Foundation Robertson Investigator. I.S., L.G., B.Z., and F.Z. are inventors on provisional patent application 62/181,453 applied for by the Broad Institute and MIT that covers the engineered CRISPR proteins described in this manuscript. Plasmid DNA encoding eSpCas9(1.0) and eSpCas9(1.1) are available from Addgene under a Universal Biological Material Transfer Agreement with the Broad Institute and MIT. F.Z. is a founder and scientific advisor for Editas Medicine and a scientific advisor for Horizon Discovery. Further information about the protocols, plasmids, and reagents can be found at the Zhang laboratory website (www.genome-engineering.org).